D. Sayer

The genetic basis for the association of the 8.1 ancestral haplotype (A1, B8, DR3) with multiple immunopathological diseases

Summary: An individuals major histocompatibility complex (MHC) ancestral haplotype (AH) is the dearest single determinant of susceptibility to MHC associated immunopathological disease, as it defines the alleles carried at all loci in the MHC. However, the direct effects of any of the 150–200 genes that constitute the MHC are difficult to determine since recombination only occurs at defined hotspots. This review concerns the 8.1 AH (HLA‐A1, C7, B8, C4AQ0, C4B1, DR3, DQ2), which is carried by most Caucasians with HLA‐B8. It is associated with accelerated human immunodeficiency virus (HIV) disease, and susceptibility to insulin‐dependent diabetes mellitns (IDDM), systemic lupus erythematosus, dermatitis herpetiformis, common variable immunodeficiency and IgA deficiency, myasthenia gravis and several other conditions. We have mapped susceptibility genes for HIV, IDDM and myasthenia gravis co the central MHC between HLA‐B and the tumour necrosis factor or complement genes. Here we consider which of the remaining 8.1‐associated diseases are more closely associated with HLA‐DR3 and/or DQ2. Several candidate genes in the central MHC have the potential to modulate immune or inflammatory responses in an antigen‐independent manner, as is seen in studies of cultured cells from healthy carriers of the 8.1 AH. Hence these genes may act as a common co‐factor in the diverse immunopathological conditions associated with the 8.1 AH.

Population frequencies and putative haplotypes of the killer cell immunoglobulin-like receptor sequences and evidence for recombination.

BACKGROUND The killer cell immunoglobulin-like receptors (KIR) are a family of receptors expressed on natural killer (NK) cells and some T cells. Class I HLA molecules on target cells are the ligands for the KIR receptors. The number of KIR genes has been reported to vary between individuals, resulting in different KIR haplotypes. There is little published data on the frequency of each KIR gene and the linkage disequilibrium between the genes. Because there is evidence that NK cells may be involved in bone marrow transplant rejection, we have determined the KIR gene frequencies and possible haplotypic arrangements by linkage disequilibrium analysis in an Australian population. METHODS Controls, patients with leukemia, and unrelated bone marrow donor-recipient pairs were typed for the presence of 11 KIR genes by polymerase chain reaction-sequence specific priming. RESULTS Ninety percent of the population was found to have a sufficient number and variety of KIR genes to detect any mismatch of HLA-A, -B, and -C alleles on NK target cells. The 11 KIR genes could be divided into two groups based on linkage disequilibrium between pairs of genes. Evidence for a recombination within the KIR gene complex is presented. CONCLUSION Typing for the presence of particular KIR genes may be indicated for bone marrow donor-recipient pairs for whom a class I HLA mismatch is unavoidable.

High‐resolution, high‐throughput HLA genotyping by next‐generation sequencing

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome. Hematopoietic stem cell transplantation requires allele-level HLA typing at multiple loci to select the best matched unrelated donors for recipient patients. In current methods for HLA typing, both alleles of a heterozygote are amplified and typed or sequenced simultaneously, often making it difficult to unambiguously determine the sequence of the two alleles. Next-generation sequencing methods clonally propagate in parallel millions of single DNA molecules, which are then also sequenced in parallel. Recently, the read lengths obtainable by one such next-generation sequencing method (454 Life Sciences, Inc.) have increased to >250 nucleotides. These clonal read lengths make possible setting the phase of the linked polymorphisms within an exon and thus the unambiguous determination of the sequence of each HLA allele. Here we demonstrate this capacity as well as show that the throughput of the system is sufficiently high to enable a complete, 7-locus HLA class I and II typing for 24 or 48 individual DNAs in a single GS FLX sequencing run. Highly multiplexed amplicon sequencing is facilitated by the use of sample-specific internal sequence tags (multiplex identification tags or MIDs) in the primers that allow pooling of samples yet maintain the ability to assign sequences to specific individuals. We have incorporated an HLA typing software application developed by Conexio Genomics (Freemantle, Australia) that assigns HLA genotypes for these 7 loci (HLA-A, -B, -C, DRB1, DQA1, DQB1, DPB1), as well as for DRB3, DRB4, and DRB5 from 454 sequence data. The potential of this HLA sequencing system to analyze chimeric mixtures is demonstrated here by the detection of a rare HLA-B allele in a mixture of two homozygous cell lines (1/100), as well as by the detection of the rare nontransmitted maternal allele present in the blood of a severe combined immunodeficiency disease syndrome (SCIDS) patient.

A multi-site study using high-resolution HLA genotyping by next generation sequencing

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

A randomised, open-label comparison of three highly active antiretroviral therapy regimens including two nucleoside analogues and indinavir for previously untreated HIV-1 infection: The OzCombo1 study

BackgroundHighly active antiretroviral therapy (HAART) including two nucleoside analogues and a potent protease inhibitor is standard of care initial therapy for HIV-infected adults. The best-tolerated and most potent initial HAART regimen is unknown and was investigated in this study. MethodsOne hundred and nine HIV-infected adults with no prior antiretroviral therapy, and CD4 lymphocyte counts < 500 × 106 cells/l or plasma HIV RNA > 30 000 copies/ml were randomized to zidovudine–lamivudine–indinavir (ZDV–3TC–IDV), stavudine–lamivudine–indinavir (d4T–3TC–IDV) or stavudine–didanosine–indinavir (d4T–ddI–IDV) for 52 weeks. The primary endpoints were plasma HIV RNA and drug-related adverse events. Other assessments were overall safety, adherence and adverse events, CD4 lymphocyte counts, cutaneous delayed type hypersensitivity (DTH) responses and quality of life (Euroqol). ResultsOnly 58% patients had HIV RNA < 50 copies/ml plasma at 12 months, with no significant difference between the three regimes (P  = 0.34). Drug-related adverse events sufficiently severe to warrant drug discontinuation were less common (P  = 0.06) in patients receiving d4T–3TC–IDV (18%) than in those receiving ZDV–3TC–IDV (34%) or d4T–ddI–IDV (41%). The percentages of patients who remained on their assigned therapy with plasma HIV RNA < 50 copies/ml at 52 weeks were 60% with d4T–3TC–IDV, 53% with ZDV–3TC–IDV and 35% with d4T–ddI–IDV. Virological failure at 52 weeks was more likely in those whose adherence was estimated to be < 100% in the first 4 weeks of therapy (P  = 0.02), but not in those who developed grade 3 or 4 drug-related adverse events. At 52 weeks, the mean CD4 lymphocyte count increase was 200 × 106 cells/l with only 7% of patients having counts lower than at baseline; DTH responses improved but remained clinically impaired in most patients. Quality of life improved significantly in all groups. ConclusionsInitial HAART regimens including IDV failed to suppress plasma HIV RNA to < 50 copies/ml in > 40% patients after only 12 months of therapy although there was significant overall improvement immunologically and in quality of life. The type of dual nucleoside combination used was less important in predicting virological failure than was imperfect adherence early in therapy. Consideration should be given to modifying a HAART regimen relatively early in non-adherent patients.

Treatment response and durability of a double protease inhibitor therapy with saquinavir and ritonavir in an observational cohort of HIV-1-infected individuals.

Objective:To evaluate treatment response, durability and tolerance of a four-drug regimen including saquinavir and ritonavir in combination with either zidovudine/lamivudine or stavudine/lamivudine. Design:Observational cohort of HIV-positive individuals. Methods:Viral load, CD4+ and CD8+ T lymphocyte counts were assessed at intervals of 1–3 months in subjects commencing therapy between July 1996 and November 1996. Adverse events were evaluated as well as risk factors for therapeutic failures. Results:A group of 56 male patients were included and followed for 48 weeks. Of these, 66% had already taken a protease inhibitor. Viral load dropped by a median 1.98 log10 HIV RNA copies/ml from baseline (interquartile range: 1.49–2.46) and became undetectable (< 400 copies/ml) in 68% of patients. Response varied: 9% were non-responders (HIV RNA reduction < 0.5 log10 copies/ml) and 23% were incomplete responders (nadir of HIV RNA > 400 copies/ml). After 48 weeks, viral load remained undetectable in 49%. Median CD4+ T lymphocyte count increased from 191 × 106 to 418 × 106 cells/l (range, 241–537 × 106 cells/l). Although protease inhibitor and nucleoside pretreatment selected for drug-resistant viral mutants, only the protease inhibitor experience was identified as a risk factor for therapeutic failure. Adverse events occurred in 73% of patients and led to a change of therapy in 9%. Conclusion:Despite advanced HIV disease and pretreatment with multiple antiretroviral drugs, a strong initial treatment response to this drug regimen was observed. However, virological failure occurred in 51% of patients after 48 weeks and frequent adverse events complicated therapy.

Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study.

BACKGROUND A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. OBJECTIVES A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project. STUDY DESIGN Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C. RESULTS Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation. CONCLUSION All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.

TREAT Asia Quality Assessment Scheme (TAQAS) to standardize the outcome of HIV genotypic resistance testing in a group of Asian laboratories.

The TREAT Asia (Therapeutics, Research, Education, and AIDS Training in Asia) Network is building capacity for Human Immunodeficiency Virus Type-1 (HIV-1) drug resistance testing in the region. The objective of the TREAT Asia Quality Assessment Scheme - designated TAQAS - is to standardize HIV-1 genotypic resistance testing (HIV genotyping) among laboratories to permit rigorous comparison of results from different clinics and testing centres. TAQAS has evaluated three panels of HIV-1-positive plasma from clinical material or low-passage, culture supernatant for up to 10 Asian laboratories. Laboratory participants used their standard protocols to perform HIV genotyping. Assessment was in comparison to a target genotype derived from all participants and the reference laboratorys result. Agreement between most participants at the edited nucleotide sequence level was high (>98%). Most participants performed to the reference laboratory standard in detection of drug resistance mutations (DRMs). However, there was variation in the detection of nucleotide mixtures (0-83%) and a significant correlation with the detection of DRMs (p<0.01). Interpretation of antiretroviral resistance showed approximately 70% agreement among participants when different interpretation systems were used but >90% agreement with a common interpretation system, within the Stanford University Drug Resistance Database. Using the principles of external quality assessment and a reference laboratory, TAQAS has demonstrated high quality HIV genotyping results from Asian laboratories.

Quality Assessment Program for Genotypic Antiretroviral Testing Improves Detection of Drug Resistance Mutations

ABSTRACT Genotypic antiretroviral testing is now widely used for the management of patients who are undergoing antiretroviral therapy for human immunodeficiency virus infection. The assays are complex, and there is considerable potential for variation between laboratories. Informative and ongoing quality assessment programs (QAPs) which address all aspects of testing are required. The panel distribution of clinical material is a critical component of QAPs. We report on the results and data from a recent panel. Four cryopreserved plasma samples from treated donors were distributed to nine laboratories. Three laboratories performed testing by commercial assays, and six laboratories used in-house assays, with one laboratory reporting results from two in-house assays. There was complete concordance between results for 95.9% of the nucleotide sequence and 94.5% of the amino acid sequence. Despite this overall high level of concordance, the degree of concordance at drug resistance mutation (DRM) sites when DRMs were present was considerably less (38% of DRM sites). Consequently, only 3 of the 10 methods reported 100% of DRMs as present. This elevated discrepancy rate is almost certainly a result of variability in the identification of mixtures of nucleotides (mixtures) at any site within the sequence. In addition, laboratories differed in the number of codons in the reverse transcriptase gene that were sequenced and their ability to amplify all samples. This panel distribution demonstrated a requirement for laboratory participation in ongoing QAPs and the optimization of assays with standards that contain mixtures.

Contributions of vitamin D response elements and HLA promoters to multiple sclerosis risk

Objective: The identification of a vitamin D–responsive (VDRE) motif within the HLA-DRB1*15:01 promoter region provides an attractive explanation for the combined effects of HLA-DR inheritance and vitamin D exposure on multiple sclerosis (MS) risk. We therefore sought to incorporate HLA-DRB1 promoter variation, including the VDRE motif, in an assessment of HLA-DRB1–associated MS risk. Methods: We utilized 32 homozygous HLA cell lines (covering 17 DRB1 alleles) and 53 heterozygote MS samples (20 DRB1 alleles) for HLA-DRB1 promoter sequencing. The influence of HLA-DRB1 variation on MS risk was then assessed among 466 MS cases and 498 controls. Results: The majority of HLA*DRB1 alleles (including HLA-DRB1*15:01) express the functional VDRE motif, apart from HLA-DRB1*04, *07, and *09 alleles that comprise the HLA-DR53 serologic group. Allele-specific variation within functional X-box and Y-box motifs was also associated with serologically defined HLA-DR haplotypes. Incorporating these results in an analysis of MS risk, we identified a strong protective effect of HLA-DRB1*04, *07, and *09 (DR53) alleles (p = 10−12) and elevated risk associated with DRB1*15 and *16 (DR51) and *08 (DR8) alleles (p < 10−18). Conclusions: HLA-DRB1 groups corresponding to serologic HLA-DR profiles as well as promoter polymorphism haplotypes effectively stratified MS risk over an 11-fold range, suggesting functional relationships between risk-modifying HLA-DRB1 alleles. An independent contribution of VDRE motif variation to increase MS risk was not discernible, although vitamin D–dependent regulation of HLA-DR expression may still play an important role given that HLA-DRB1*04/*07/*09 (DR53) alleles that express the “nonresponsive” VDRE motif were associated with significantly reduced risk of MS.